It is very important to elucidate the mechanism of increased intraocular pressure in glaucoma and to control postoperative fibrosis. The research has progressed; however, the details are still unclear. In this study, we describe new results from our research and the possibility of novel therapies.
I. Pathophysiology of increased aqueous outflow resistance in the main pathway
Intraocular pressure (IOP) is controlled by the balance between the production and drainage of aqueous humor. Increased IOP in glaucomatous eyes is attributed to increased aqueous outflow resistance in the primary outflow pathway. It has been reported that bioactive substances in aqueous humor are involved in increased outflow resistance because of the fibrosis of the angle trabecular meshwork (TM), abnormal deposition of the extracellular matrix (ECM), and enhanced barrier function in Schlemm's endothelial cells. In particular, transforming growth factor beta 2 (TGFβ2), a typical cytokine involved in fibrosis, has been reported to be high in the aqueous humor of primary open-angle glaucoma (POAG), but low in the aqueous humor of secondary glaucoma (SOAG) and exfoliation glaucoma (XFG), which are frequently associated with very high IOP and IOP fluctuation. The factors associated with increased IOP in these disease types were unclear.
II. ATX-LPA pathway in the pathophysiology of glaucoma
We analyzed the aqueous humor of numerous patients with various types of glaucoma, focusing on lysophosphatidic acid (LPA), which is a lipid mediator involved in fibrosis and has been reported to contribute to an increase in outflow resistance of the aqueous humor, and the production of enzyme autotaxin (ATX). The results demonstrated that LPA and ATX were significantly increased in eyes with glaucoma, particularly with SOAG and XFG, which were significantly correlated with IOP. Furthermore, the analyses of human angle tissues and cultured human trabecular cells demonstrated that expression of ATX increased in the TM of glaucomatous eyes and that increased ATX expression in cultured human trabecular cells upon steroid stimulation was associated with fibrosis and inhibited by Rho-associated protein kinase (ROCK) inhibitors. Thus, in vitro studies were conducted using cultured human trabecular cells and cultured Schlemm's endothelial cells to investigate the effects of ATX and LPA on the aqueous humor outflow tract and compare the effects of TGFβ1 and TGFβ2, which have been reported to be involved in the increased resistance of the main pathway in previous studies. Cytoskeletal changes and increased ECM expression were observed in cultured human trabecular cells treated with ATX and LPA. Moreover, increased cell adhesion and increased barrier function were observed in cultured Schlemm's endothelial cells. Thus, the ATX-LPA pathway was confirmed to be involved in increased outflow resistance of the primary pathway. LPA was reported to have a more rapid onset of action than ATX. The immediate effect of TGFβ1 on the primary pathway was weaker than that of TGFβ2, and the onset of action of TGFβ2 tended to be more delayed than that for ATX and LPA. Significant increases in IOP were observed 0.5-1.5 h, 1-2 h and 24 h after instillation of LPA and ATX and TGFβ2, respectively, indicating differences in time to exert significant effects on the increase in IOP between them.
III. ATX-LPA pathway in secondary glaucoma
Next, the involvement of the ATX-LPA pathway in the pathophysiology of secondary glaucoma was investigated. Posner-Schlossman syndrome (PSS) is particularly possible to evolve to refractory glaucoma in cytomegalovirus (CMV) -positive patients because of persistent paroxysmal ocular hypertension. The results of aqueous humor analyses demonstrated there was a significant correlation between elevated values of ATX and increased IOP in PSS patients with concurrent glaucoma. Furthermore, in an in vitro pathological model of CMV infection using cultured human trabecular cells and cultured Schlemm's endothelial cells, increased expression of ATX and TGFβ1, decreased TGFβ2, fibrosis of cultured human trabecular cells, and enhanced barrier function of cultured Schlemm's endothelial cells were observed.
These results suggested that the ATX-LPA pathway is involved in the pathophysiology of secondary glaucoma and that the balance between ATX and TGFβ expression may differ as per the IOP level and disease type. Thus, aqueous humor analyses were performed in a group of newly diagnosed patients. The results demonstrated that the balance between ATX and TGFβ was reflected by higher area under the curve (AUC) for either one of two factors as per the diagnosis of glaucoma type, particularly higher AUC for ATX in the differential diagnosis of XFG, and was considered to be a possible biomarker for the type of glaucoma. Clinical utility will be evaluated further in future.
IV. Fibrosis after glaucoma surgery and lipid mediators
Moreover, we considered that ATX in the aqueous humor may affect the increase in IOP and fibrosis after glaucoma surgery, as well as investigated the relationship between the maintenance of filtration blebs after filtration surgery, ATX and disease type, in addition to the effect of postoperative steroid instillation in combined ab interno trabeculotomy and cataract surgery. After filtration surgery, a significant post-operative bleb fibrosis was observed in XFG with high ATX levels. Multivariate analysis revealed that ATX was a factor significantly correlated with the requirement for needling. In cultured Tenon's fibroblasts, ATX treatment induced fibrosis and increased ECM expression, which were inhibited by ATX and ROCK inhibitors. In combined ab interno trabeculotomy and cataract surgery, the number of postoperative instillations was significantly larger in the steroid combination group, and the postoperative change in ATX levels was significantly correlated with the difference between the preoperative IOP and the postoperative IOP at 3 months after surgery. In cultured human trabecular cells, dexamethasone-induced fibrosis and increased ECM expression were enhanced by adding stretch stimulation and inhibited by ROCK inhibitors.
V. Possibility of discovering novel drugs targeting the ATX-LPA pathway
The results suggest that ATX-LPA pathway targeted therapy may be useful for both reducing IOP and improving postoperative results. Thus, we performed drug screening and optimization using the University of Tokyo Drug Discovery Initiative Compound Library with the aim of developing a new drug, as well as created a new highly selective ATX inhibitor, which was confirmed to reduce IOP in animal models of increased IOP.
In future, the elucidation of the pathogenesis of increased IOP mediated by aqueous humor factors and the development of new therapeutic methods through discovery of new drugs to control increased IOP is expected. Our research suggested that glaucoma treatment targeting the ATX-LPA pathway may be useful.
Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 125: 285-322, 2021.